Trypsin inhibitors in guinea pig plasma: isolation and characterization of contrapsin and two isoforms of alpha-1-antiproteinase and acute phase response of four major trypsin inhibitors

Contrapsin and two isoforms, F (fast) and S (slow), of alpha-1-antiproteinase (also called alpha-1-proteinase inhibitor) were isolated in an apparently homogeneous state from plasma of inflamed guinea pigs. Contrapsin inactivated trypsin, but did not significantly affect chymotrypsin, pancreatic elastase, or pancreatic kallikrein. On the other hand, both isoforms of alpha-1-antiproteinase inhibited trypsin, chymotrypsin, and elastase, but not plasma or pancreatic kallikrein. The S isoform of alpha-1-antiproteinase was present in barely detectable amounts in healthy animals, but increased markedly when the acute-phase reaction was induced by subcutaneous injection of turpentine. On the other hand, the plasma levels of the F isoform, contrapsin, and alpha-macroglobulin showed moderate (1.5 to 2.3-fold) elevation during the acute-phase reaction. In contrast to the previous findings that rats and rabbits contain two different alpha-macroglobulins, one of which is an acute-phase reactant while the other is not, inflamed guinea pigs contained only one species of alpha-macroglobulin. Murinoglobulin, the most prominent acute-phase negative protein in both mice and rats, showed no significant change in guinea pigs. These results indicate that guinea pig plasma contains four major trypsin inhibitors, i.e., contrapsin, alpha-1-antiproteinase, alpha-macroglobulin, and murinoglobulin, the properties of which are very similar to those of the respective mouse homologues, but that the acute-phase response of these inhibitors differs greatly from that of the homologous proteins in rats or mice.     

Murinoglobulin, a novel protease inhibitor from murine plasma. Isolation, characterization, and comparison with murine alpha-macroglobulin and human alpha-2-macroglobulin

Two glycoproteins having trypsin-protein esterase activity were purified to apparent homogeneity from murine plasma. One was alpha-macroglobulin, a homologue of human alpha-2-macroglobulin, while the other, tentatively named murinoglobulin, did not correspond to any of the known plasma protease inhibitors that have been well characterized in men or other mammals. Murinoglobulin contained about 7.6% carbohydrate and was composed of a single-polypeptide chain of Mr = 180,000 as judged by the equilibrium sedimentation analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Murinoglobulin did not cross-react immunologically with mouse alpha-macroglobulin nor with human alpha-2-macroglobulin. Protease-inhibiting properties of murinoglobulin were compared with those of mouse alpha-macroglobulin and human alpha-2-macroglobulin. All the three proteins inhibited trypsin, papain, and thermolysin, although they differed considerably in both the degree of inhibition and the binding stoichiometry of protease-inhibitor complexes. The two macroglobulins inhibited pepsin at pH 5.5, whereas murinoglobulin was inactivated at this pH. Murinoglobulin was more sensitive to methylamine than the two macroglobulins. No protein corresponding to murinoglobulin was detected in human plasma.     

Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.     

Clearance of desialylated mouse alpha-macroglobulin and murinoglobulin in the mouse.

Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver.     

Rat plasma murinoglobulin: isolation, characterization, and comparison with rat alpha-1- and alpha-2-macroglobulins

Murinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1 mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr = 210,000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat alpha-1-macroglobulin but not towards rat or human alpha-2-macroglobulin. The chemical compositions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat alpha-1- or alpha-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two alpha-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murinoglobulin, but not the alpha-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two alpha-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two alpha-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two alpha-macroglobulins.     

Inhibitory spectrum of mouse contrapsin and alpha-1-antitrypsin against mouse serine proteases

Contrapsin and alpha-1-antitrypsin have been recently characterized as major protease inhibitors in mouse plasma (Takahara, H. & Sinohara, H. (1982) J. Biol. Chem. 257, 2438-2446). We have studied the effects of the two inhibitors upon various serine proteases prepared from mouse tissues. Trypsin, plasmin and trypsin-like proteases of the submaxillary gland were inhibited by contrapsin but not by alpha-1-antitrypsin. On the other hand, chymotrypsin, elastase, and thrombin were inactivated by alpha-1-antitrypsin but not by contrapsin. Thus, their inhibitory spectra did not overlap each other in spite of their broad specificities. The inhibition of trypsin, chymotrypsin, and elastase was rapid and stoichiometric, whereas the inhibition of the other proteases was relatively slow. Contrapsin accounted for almost the total capacities of mouse plasma to inhibit both trypsin and submaxillary gland trypsin-like proteases, whereas alpha-1-antitrypsin was responsible for nearly all the capacities of plasma to inhibit both chymotrypsin and elastase.     

Synthesis of contrapsin and alpha-1-antiproteinase in inflamed and tumor-bearing mice

In mice bearing ascites tumors, such as Ehrlich, L1210, Meth-A, and P-815, the plasma levels of contrapsin and alpha-1-antiproteinase remained virtually unchanged. However, the total body pools of these proteins as well as their hepatic mRNA levels increased severalfold, and isoelectrofocusing patterns of these proteins shifted to the lower pH side. Under the same conditions, plasma albumin level decreased by 25%, but its total body pool and hepatic mRNA level increased severalfold. On the other hand, induction of acute phase reaction by injection of bacterial lipopolysaccharide caused differential effects on the two proteinase inhibitors: hepatic translatable mRNA for contrapsin was doubled while that for alpha-1-antiproteinase remained unchanged.     

Isolation and characterization of alpha-macroglobulin from guinea pig plasma

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.     

Amidolytic activity of porcine trypsin bound to human plasma alpha-1-antiproteinase

Human plasma alpha-1-antiproteinase interacted with porcine trypsin in two different manners. One was a well known interaction, which resulted in inhibition of the proteolytic activity of the trypsin. The other has not been described to date, and resulted in retention of the amidolytic activity of the trypsin towards benzoyl-L-arginine p-nitroanilide in the presence of soybean trypsin inhibitor. The latter, so-called trypsin-protein amidase, activity is essentially the same as that observed with vertebrate alpha-macroglobulin and rodent murinoglobulin under similar conditions. All attempts to separate the two different activities as well as to abolish either activity by means of chemical or physical modifications were unsuccessful. The proteolysis-inhibiting interaction, which was virtually completed within 5 min, was predominant over the amidolysis-retaining interaction, when the inhibitor/trypsin molar ratio was less than 1. On the other hand, the amidolysis-retaining interaction, which proceeded much more slowly, became evident when the molar ratio was greater than 1.     

Molecular cloning and sequence analysis of full-length cDNA coding for mouse contrapsin

Four overlapping cDNA clones encoding contrapsin were isolated from a mouse liver cDNA library constructed in the expression vector, lambda gt11. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1,254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human alpha-1-antichymotrypsin.     

Regulation of acute phase reaction by transforming growth factor beta in cultured murine hepatocytes.

Transforming growth factor-beta (TGF beta 1), a multipotent immunoregulatory peptide produced by human platelets, has been shown to stimulate the synthesis of fibrinogen, contrapsin, complement component C3, and alpha-1-proteinase inhibitor by murine hepatocytes cultured for 2 days in DMEM containing 1 microM insulin and dexamethasone and 0.2% BSA. In the range of 10 pg to 10 ng/ml TGF-beta 1 did not elicit any change in albumin secretion. Two main inflammatory cytokines: interleukin-6 (IL-6) and interleukin-1 (IL-1), known to stimulate two different subsets of murine acute phase plasma proteins, failed to increase contrapsin and alpha-1-proteinase inhibitor production. Epidermal growth factor (EGF) in the concentration 1 ng to 10 ng/ml effectively counteracted the stimulatory effect of TGF-beta 1 on acute phase protein production. TGF-beta 1-induced fibrinogen protein levels were associated with increased beta-fibrinogen mRNA content. TGF-beta 1 appears to be an additional physiological factor responsible for the direct stimulation of normal mouse hepatocytes to acute phase response.     

New Anthracycline Antibiotic CG12 Obtained by Microbial Glycosidation of α-Citromycinone

We studied the effects of peptidylarginine deiminase, a Ca²⁺-dependent protein-modulating enzyme that catalyzes the deimination of arginyl residues in protein on two major trypsin inhibitors in mouse plasma. One of these inhibitors was α-1-antitrypsin and the other was a recently characterized trypsin inhibitor termed contrapsin (H. Takahara and H. Sinohara, J. Biol. Chem., 257, 2438 (1982)). The enzyme abolished the trypsin-inhibiting activity of contrapsin in a process that was pseudo-first order with the rate dependent on enzyme concentration (second order rate constant = 3.4 × 10³ m^-1 · s^-1), but no detectable changes in the activity was noted for α-1-antitrypsin. Millimolar Ca²⁺ and dithiothreitol were absolutely required for the inactivation of contrapsin by peptidylarginine deiminase. Although no significant alterations of the charge and the size in modified contrapsin were observed, the modified inhibitor indicated that 1 arginyl residue was converted to a citrullyl residue. Modified contrapsin whose anti-tryptic activity was lost inhibited chymotrypsin much more strongly than the native inhibitor. These data suggest that a vital amino acid residue for the anti-tryptic activity of mouse contrapsin is an arginyl residue and the conversion of this arginyl residue to a citrullyl residue causes the functional changes of the inhibitor.     

Mouse alpha 1-protease inhibitor is not an acute phase reactant

Mouse plasma contains two major protease inhibitors, alpha 1-protease inhibitor (alpha 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of alpha 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic alpha 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J.M. Frazer, S.A. Nathoo, J. Katz, T.L. Genetta, and T.H. Finley [1985) Arch. Biochem. Biophys. 239, 112-119) have reported a threefold increase of mRNA for the elastase specific alpha 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, alpha 1-PI is not an acute phase reactant.     

Cell coupling and maturation-promoting factor activity in in vitro-matured prepubertal and adult sheep oocytes.

We examined some differences between prepubertal and adult ovine oocytes; in particular we analyzed the functional status of the cumulus-oocyte complex, protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, maturation-promoting factor (MPF) fluctuations throughout meiotic progression both in prepubertal and adult sheep oocytes. After 24 h of maturation, percentages of MII oocytes were similar between prepubertal and adult animals. Electron microscopy examinations showed that prepubertal oocytes had fewer transzonal projections than adult oocytes. Methionine uptake was significantly lower in prepubertal cumulus-enclosed oocytes examined through meiotic progression. On the contrary, denuded prepubertal oocytes showed a higher methionine incorporation in the first 4 h of incubation compared with adult oocytes. We also found some differences in MPF activity between prepubertal and adult oocytes at MII stage. In fact, prepubertal MII oocytes had a significantly lower level of MPF activity than adult oocytes did and, after fusion with germinal vesicle oocytes, they were unable to induce nuclear breakdown and chromosome condensation 1-2 h post-fusion, whereas adult MII oocytes could induce these processes. Our findings show that the lesser competence of prepubertal oocytes could be due to morphological anomalies and alterations in physiological activity and that oocytes do not reach full developmental competence until puberty.     

Acute phase response of plasma proteins in analbuminemic rats

In healthy Nagase analbuminemic rats (NAR), the highest degree of relative increase in serum protein concentration was found for alpha-2-macroglobulin, the most prominent acute phase protein in rats. Its levels were about 30- and 60-fold higher in males and females, respectively, than those in the control Sprague-Dawley (SD) rats. In terms of absolute concentration, however, alpha-1-inhibitor 3 (also called alpha-X-protein or murinoglobulin) showed the most conspicuous change, its levels being higher by about 7 mg/ml than those in SD. When the acute phase reaction was induced by subcutaneous injection of turpentine, the levels of alpha-1- and alpha-2-macroglobulins, alpha-1-cysteine proteinase inhibitor, alpha-1-antiproteinase, and alpha-1-inhibitor 3 in NAR changed in essentially the same way as in SD: alpha-1-inhibitor 3 decreased markedly while the rest increased further. These results suggest that mechanisms responsible for the elevation of serum globulins in healthy NAR are not directly related to those involved in the acute phase response. On the other hand, the antithrombin III levels in healthy NAR were about twice the control values and changed little during the inflammation. In contrast, this protein in SD doubled during the acute phase, its maximal levels being close to those in healthy or inflamed NAR. This suggests that the antithrombin III level in healthy NAR is regulated by a mechanism similar to that in SD maximally reacting to the acute phase stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications of serum glycoproteins the days following a prolonged physical exercise and the influence of physical training.

Eight male subjects (mean age 24.1 +/- 2.6 years) performed at intervals of 2 weeks successively a 3 h and two 2 h runs of different running speed. The days following the running there were moderate elevations of C-reactive protein, haptoglobin, alpha-1-acid glycoprotein, coeruloplasmin, transferrin, alpha-1-antitrypsin and plasminogen. There were small or no changes of albumin, alpha-2-macroglobulin and hemopexin. The elevations of the "acute phase reactants" were examined in three male subjects following a 2 h run before and after an endurance training period of 9 weeks. This demonstrated a decreased acute phase response after training as illustrated by the changes of C-reactive protein, haptoglobin and alpha-1-acid glycoprotein in spite of higher posttraining running speeds. Well-trained athletes have elevated levels of the serum protease inhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and C1-inhibitor. These antiproteolytic glycoproteins might limit exercise-induced inflammatory reactions.     

Identification of Four Genes Coding for Isoforms of Murinoglobulin, the Monomeric Mouse α2-Macroglobulin: Characterization of the Exons Coding for the Bait Region

Murinoglobulins are the single chain members of the α₂-macroglobulin family of proteinase inhibitors in the mouse. DNA clones representing the genes coding for four different murinoglobulins were isolated from three independent mouse genomic DNA libraries. Sequence analysis demonstrated that in each gene two exons are coding for the bait region. This is the specific protein sequence in each α-macroglobulin, which is functionally important since it is extremely sensitive to cleavage by different proteinases. The molecular data established the existence of at least four different murinoglobulin genes. Three of these corresponded to the three cDNA clones previously identified. Sequencing of intron-exon boundaries and intron sizing allowed us to construct physical maps of the region from exon 15 to exon 25 (numbered in comparison to mouse α₂-macroglobulin) in each murinoglobulin gene. Southern blotting of genomic DNA from five different mouse strains confirmed this analysis and even suggested the possible existence of a fifth murinoglobulin gene. These data indicate that the mouse presents genetic repertoire of the α₂-macroglobulin family much more complex than originally anticipated. The bait region exon sequences showed a considerably higher degree of divergence (72 to 88% sequence identity) than that of the flanking exon sequences coding for adjacent, structural domains of the murinoglobulin proteinase inhibitors (91 to 96%). Even more surprising was that adjacent intron sequences are conserved as faithfully as the nonbait region coding exons (90 to 96%). These data demonstrate a unique property of the bait region coding sequences, as they apparently are allowed to mutate considerably. This divergency must then confer divergent proteinase inhibitory properties to the resulting proteins.     

Reference maps of mouse serum acute-phase proteins: changes with LPS-induced inflammation and apolipoprotein A-I and A-II transgenes

We present reference maps of the mouse serum proteome (run under reducing and non-reducing conditions), from control animals, from mice injected with lipopolysaccharide (LPS) to induce systemic inflammation, and from mice transgenic for human apolipoproteins A-I and A-II. Seventy-seven spots/spot chains from the reducing gels were identified by HPLC MS/MS, representing 28 distinct proteins, including a species-specific protease inhibitor, contrapsin, and high levels of carboxylesterase. The concentrations of acute-phase reactants were monitored for 96 h after LPS challenge. The greatest changes (four-fold 48 h after LPS administration) were observed for haptoglobin and hemopexin. Orosomucoid/alpha(1)-acid glycoprotein and apolipoprotein A-I increased steadily, to 50-60% above baseline at 96 h from stimulation. In mice transgenic for human apolipoprotein A-I the levels of expression of orosomucoid/alpha(1)-acid glycoprotein, alpha(1)-macroglobulin, esterase, kininogen and contrapsin were altered compared to knockout mice lacking apolipoprotein A-I. In contrast, except for the presence of apolipoprotein A-II, no statistically significant difference was observed in mice transgenic for human apolipoprotein A-II.     

Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes

The aim of this study was to analyze different culture systems on embryo development of prepubertal goat oocytes. We compare (i) the effect of the age of donor (goat) of oocytes on in vitro maturation, fertilization and subsequent embryo development, (ii) the effect of the origin of oviduct cells from coculture of prepubertal goat embryo development, and (iii) the effect of in vivo culture in rabbit oviducts for 1, 2 and 3 days on the development of prepubertal goat embryos produced in vitro. In Experiment 1, at 24 h post-insemination (hpi), oocytes from adult goats were allocated in TCM199 with oviduct cells from adult goats, and oocytes from prepubertal goats were randomly placed in drops with oviduct epithelial cells from adult (aOEC) or prepubertal (pOEC) goats. Cleavage rate and embryo development were evaluated at 48 hpi and after 7 days coculture, respectively. In Experiment 2, at 24 hpi, prepubertal oocytes were allocated in TCM 199 with pOEC. At 40-42 hpi, a group of embryos remained in the coculture (control group), and the rest were transferred to rabbit oviducts (three rabbits for replicate) for culturing in vivo for 24, 48 and 72 h. After these in vivo cultures, embryos were recovered, evaluated and placed in TCM199 with pOEC until Day 8 post-insemination. The maturation, fertilization and blastocyst rates did not differ significantly between oocytes obtained from adult and prepubertal goats. The percentage of blastocysts obtained from prepubertal goat embryos cocultured with aOEC or pOEC was also similar (12.1% versus 12.2%). The transfer of prepubertal goat embryos to rabbit oviducts for 1, 2 and 3 days did not improve the blastocyst rate compared to the control group (9.7, 10.9, 4.1 and 11.5%, respectively). In conclusion, in our conditions, there were no significant differences in embryo development between oocytes obtained from prepubertal and adult goats, and the embryo development from prepubertal goat oocytes were similar in the different culture systems compared.